Formulation

ABSTRACT

The present invention relates to a new formulation of the oligonucleotide of SEQ ID NO: 1.

BACKGROUND OF THE INVENTION 1. The Field of the Invention

The present invention relates to a new formulation of theoligonucleotide of SEQ ID NO: 1.

2. The Relevant Technology

Mucosal surfaces represent the first interface between the internalenvironment of the host organism and the external environment. Mucosalsurfaces are therefore enriched in a variety of cellular and acellularstructures that protect the host from foreign pathogen or antigenexposure. These important surfaces are represented by the mucosalsurfaces of the oral and nasal (sinus) cavity, the pulmonary anddigestive systems and mucosal tissues surrounding the eye.

The cells comprising the mucosa are equipped to sense and respond to avariety of foreign substances. These cells also elaborate a variety ofmolecular pathways to communicate such invasion to the surroundingtissues and recruit an influx of additional inflammatory and immunecells to fight the infection, repair damage and if necessary inducespecific immune responses in the form of antibodies. While thesemechanisms serve an important and indeed life-saving function for theorganism, their chronic and/or misguided activation can lead toconsiderable morbidity and mortality.

The chronic influx of inflammatory cells and the subsequent localactivation of cells of the immune system are two primary loci wherechronic mucosal inflammation can be controlled. These responses are partof the “innate immune system”. The first responding cells of the innateimmune system, such as dendritic cells and macrophages, ingest pathogensand release cytokines drawing secondary, active and defensive cells fromthe blood. These secondary invading cells must be “drawn to” the site ofinflammation by molecules on the surface of cells lining the local bloodvessels (vascular endothelium). Such molecules are themselves expressedin response to cytokines released at the initial pathogen invasion site.These molecules bind to receptors on circulating blood cells and allowlocal adhesion and subsequent diapedesis into the invasion site. Theadhesion molecules are known as intracellular adhesion molecules (ICAM)and a variety of these have been discovered. Blocking the expressionand/or function of the various ICAM's has led to the development ofseveral therapeutic products to suppress inflammatory diseases.

Cells of the innate immune system also have a well-developed repertoireof surface receptors that sense and bind microbial components expressedby bacteria, viruses, fungi and other pathogens. These receptors havebeen termed “toll-like receptors” (TLR's) and as many as 12 members ofthis family are now known in the human genome. Pathogen-encoded TLRligands fall into three broad categories: lipids and lipopeptides(TLR2/1; TLR2/6 and TLR4), proteins (TLR5 and TLR11) and nucleic acids(TLR3, 7, 8 and 9). Therapeutic targeting of certain TLR's has beenexploited as a means to stimulate the immune system (vaccine production)and agents targeting other TLR's are being developed to inhibit certainimmune functions.

An ideal therapeutic agent to target and control mucosal inflammationwould therefore be an agent with both acute and long-lasting effects onmucosa inflammation and may indeed be disease altering.

SUMMARY OF THE INVENTION

The present invention addresses this.

The present invention relates to, as a first aspect, a pharmaceuticalcomposition comprising SEQ ID NO:1 with a cation optionally selectedfrom the following list Na⁺, K⁺, Mg⁺, Ca⁺⁺, Ba⁺⁺, Mn⁺⁺, Ni⁺⁺, Li⁺⁺, Zn⁺⁺and Cr⁺⁺⁺, preferably Na⁺, more preferably 40-200 mM Na⁺, optionallyincluding 2-20 mM Mg²⁺. The concentration range is preferably 40-200 mMfor Na⁺ or K⁺, and 2-20 mM for Mg⁺⁺, Ca⁺⁺, Ba⁺⁺, Mn⁺⁺, Ni⁺⁺, Li⁺⁺, Zn⁺⁺or Cr⁺⁺⁺. For example, the pharmaceutical composition may have acombination of 40-200 mM Na⁺ and 2-20 mM Mg²⁺, or 40-200 mM Na⁺ and40-200 mM K⁺. For example the composition may comprise a combination ofNa⁺ and K⁺, or Na⁺ and Mg⁺⁺ or Na⁺ and Ca⁺⁺ or Na⁺ and Ba⁺⁺ or Na⁺ andMn⁺⁺ or Na⁺ and Ni⁺⁺ or Na⁺ and Li′ or Na⁺ and Zn⁺⁺ or Na⁺ and Cr⁺⁺⁺, orK⁺ and Mg⁺⁺ or K⁺ and Ca⁺⁺ or K⁺ and Ba⁺⁺ or K⁺ and Mn⁺⁺ or K⁺ and Ni⁺⁺or K⁺ and Li′ or K⁺ and Zn⁺⁺ or K⁺ and Cr⁺⁺⁺.

The composition of the first aspect may also comprise one or more ofhydroxymethyl cellulose, methyl paraben sodium, propylparaben sodium,monobasic sodium phosphate monohydrate, sodium hydroxide, hydrochloricacid and/or water.

The composition may be in the form of a liquid syrup, gel, film, cream,powder, tablet, enema and/or particulate, preferably which is suitablefor inhalation.

The composition of the invention may be formulated as a gel, cream,lotion, solution, suspension, emulsion, ointment, powder or particulatewhich is suitable for inhalation, tablet, spray, aerosol, foam, salve,microparticle, nanoparticle, or bioadhesive, and may be prepared so asto contain liposomes, micelles and/or microspheres.

The composition may have the components in the following ranges

SEQ ID NO: 1 4 mg hydroxymethyl cellulose 7-8 mg, optionally 7.5 mgmethylparaben sodium 16.6 mM 2.8-3.0 mg propylparaben sodium 1.4 mM0.28-3 mg monobasic sodium phosphate 4.4-4.6 mg monohydrate37.5 mMwherein the ranges given are per ml with a total of 60 ml per dose.Thus, in a treatment of 240 mg in 60 ml, the treatment is 4 mg/ml. Theabove specific dose may be a liquid enema formulation.

A second aspect of the invention relates to the composition of the firstaspect for use in medicine. The medicine may be human or veterinary.Veterinary medicine includes any animal including production and/or petanimals including, in particular dogs, cats and/or equine animals.

According to the second aspect of the invention, the medicine may be forthe prevention or treatment of inflammatory bowel disease, rectal stumpdisease, radiation-induced proctitis, pouchitis, asthma, inflammation ofthe eye, dry eye, rhinitis or sinusitis or graft versus host disease.

A third aspect of the invention is the use of SEQ ID NO:1 in themanufacture of a medicament according to the first aspect of theinvention for the prevention or treatment of inflammatory bowel disease,rectal stump disease, radiation-induced proctitis, pouchitis, asthma,inflammation of the eye, dry eye, graft versus host disease (GVHD),rhinitis or sinusitis.

All features of the first aspect of the invention also apply to thethird aspect.

A fourth aspect of the invention relates to a method of treatinginflammatory bowel disease, rectal stump disease, radiation-inducedproctitis, pouchitis, asthma, inflammation of the eye, dry eye,rhinitis, sinusitis, or grant-versus host disease, the method comprisingadministration of a composition of the first aspect of the invention toa patient in need thereof.

All features of the first aspect of the invention also apply to thefourth aspect. The patient may be a human (adult or youth) or an animal.

A fifth aspect of the invention relates to a method of making acomposition of the first aspect of the invention, the method comprisingcombining SEQ ID NO:1 with a cation, optionally selected from thefollowing list Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, Ba⁺⁺, Mn⁺⁺, Ni⁺⁺, Li⁺⁺, Zn⁺⁺, andCr⁺⁺⁺, preferably Na⁺, more preferably 40-200 mM Na⁺, optionallyincluding 2-20 mM Mg²⁺.

Methods for making this new composition are standard methods as known inthe art.

The invention provides a composition, according to the first aspect ofthe invention, as claimed in any one of claims 1 to 6, wherein thecomposition is in the form of an enema and wherein the composition isformulated in a dosage form to provide a concentration of SEQ ID NO:1 at4 mg/ml per day.

The composition may be in the form of an enema and wherein thecomposition is formulated in a dosage form to provide a concentration ofSEQ ID NO:1 at 0.25-4 mg/ml per day.

SEQ ID NO:1 is as follows: 5′-gcccaagctg gcatccgtca-3′.

The antisense oligonucleotide SEQ ID NO:1 is also known as alicaforsen.

Certain enhancements to the formulation containing the oligonucleotidesof SEQ ID NO:1 may also be necessary to aid in the retention of theactive ingredient at the site of application. For example, theformulation can be prepared using components that cause the formulationto be a liquid at room temperature, but solidify to a gel state at bodytemperature. In other instances, the formulation may be prepared as aliquid and applied to a mucosal surface, such as the nasal mucosa,followed by the application of an inert dry powder, such as methylcellulose, to retain the formulation at the site of application. Inother instances, a dry powder formulation of the composition may bemixed with the inert dry powder and applied together at the mucosa site.

The oligonucleotides SEQ ID NO:1 in accordance with this inventionpreferably comprises from about 20 to about 80 nucleic acid base units.It is more preferred that such oligonucleotides comprise from about 20to 50 nucleic acid base units, still more preferred to have from about20 to 30 nucleic acid base units, and most preferred to have from about20 to 22 nucleic acid base units. As will be appreciated, a nucleic acidbase unit is a base-sugar combination suitably bound to an adjacentnucleic acid base unit through phosphodiester or other bonds. Oneskilled in the art will understand that about 20 to about 80 nucleicacid base units includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleobase units.

In a further embodiment, the composition comprises a fragment of SEQ IDNO:1, wherein the fragment is at least 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 or 19 nucleotides in length. The fragment can hybridise to asequence in the 3′-untranslated region of the human ICAM-1 mRNA. Thefragment can hybridise under moderate or stringent conditions withnucleotides ‘cctgacg gatgccagct tgg’ (SEQ ID NO:2). Fragments include‘cccaagctg gcatccgtca’ (SEQ ID NO:3), ‘gcccaagctg gcatccgtc’ (SEQ IDNO:4) and ‘gcccaagctg gca’ (SEQ ID NO:5).

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridisable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

As herein defined, “Stringent conditions” or “highly stringentconditions”, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 [mu]g/ml), 0.1% SDS, and 10%dextran sulphate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulphate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

As used herein, conditions of moderate or high stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989).

The oligonucleotide can be modified to comprise at least onephosphorothioate linkage. Phosphorothioate modification of theoligonucleotide, by substituting a sulfur molecule for a non-bridgingoxygen molecule in each phosphodiester linkage, significantly increasesexonuclease resistance relative to unmodified DNA and prolongs the drughalf-life (Geary et al., Anti-Cancer Drug Design, 12:383-94, 1997).Phosphorothioate oligonucleotides are only minimally antigenic,non-cytotoxic and well tolerated, and their pharmacokinetic andpharmacodynamic properties are well characterized (see e.g., Butler etal., Lab. Invest, 77:379-88, 1997; Mirabelli et al., Anti-Cancer DrugDes., 6:647-61, 1991). In addition to phosphorothioate backbonemodifications, a number of other possible backbone, sugar and othermodifications are well known to those skilled in the art.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g., decisions on dosage etc., isultimately within the responsibility and at the discretion of generalpractitioners and other medical doctors, and typically takes account ofthe disorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners.

For example, in one embodiment, a suitable dose may be 2 mg/ml per dayfor example, per enema.

In another embodiment, a minimum dose would be 0.25 mg/mL per day forexample, per enema.

The composition may be administered once, twice, three or four times aday or periodically.

The composition can be administered for 2, 3, 4, 5, 6, 7, 8 or moreweeks.

For example, in one embodiment, a suitable dose per treatment may be0.05 mg to 400 mg. A treatment can be administered from once to eighttimes per day.

A suitable dose concentration may be 0.5 mg/mL to 10 mg/mL. A suitabledose administered to asthma patients may be between 0.5 to 5 ml, inparticular around 1 mL. A suitable dose administered to dry eye orinflammation of the eye patients may be between 1-100 ul, in particulararound 50 ul. A suitable enema administered dose may be between 10 to100 mL, in particular around 60 mL.

The composition may be administered once, twice, three, four, five, six,seven or eight times a day or periodically.

The composition can be administered for 1, 2, 3, 4, 5, 6, 7, 8 or moreweeks.

The composition can be administered for 1, 2, 3, 4, 5 or 6 days.

The composition may be in respect of existing aggravation, inflammation,pain and/or discharge of the effected site or may be prophylactic(preventative treatment). Treatment may include curative, alleviation orprophylactic effects.

Specific examples of diseases treatable by the invention include themucosal inflammatory conditions described below. Each disease requiresthe influx of certain blood borne inflammatory cells and activation ofimmune cells that have localized to the site of inflammation. Suchdiseases are therefore ideally treated by an agent that can block boththe influx of inflammatory cells and the activation of the innate immunesystem, as provided by the present invention.

Inflammatory bowel diseases include ulcerative colitis (UC) and Crohn'sdisease (CD), both of which are inflammatory disorders of the intestinalmucosa. Ulcerative colitis is confined to the large intestine, whileCrohn's disease can involve any region of the intestinal mucosa,including the oral cavity. Both UC and CD are characterized by theinflux of inflammatory and immune cells in response to environmental andautoimmune stimuli. Although the histopathology of the two diseasesdiffer, many therapeutic agents are used to treat both conditions, suchas steroids, anti-TNF antibodies and mesalamine.

Pouchitis is inflammation of the distal intestinal mucosa remainingafter surgical removal of the colon and formation of a J-pouch byileal-anal anastomosis. Mucosal inflammation in pouchitis is similar tothat observed in ulcerative colitis, although it may have microbialinvolvement to a greater extant. It is characterized by influx ofactivated inflammatory cells and activation of the local immune system.

Rectal stump disease is an inflammatory condition affecting the rectalmucosa remaining after colectomy.

Asthma is a complex and multifactorial disorder typified by episodes ofbreathlessness and wheezing in concert with airway hyper-reactivity(AHR) to a range of stimuli. Chronic inflammation mucosa of the airwaywall is thought to be the primary factor driving asthmaticexacerbations. It is now well established that polarized CD4 T helper 2(Th2) cells infiltrate and accumulate in the bronchial mucosa ofallergic asthmatics, and that cytokines secreted from these cells(interleukin-(IL)-4, IL-5, IL-9 and IL-13) are largely responsible foracute exacerbations and promotion of the pathologic features of allergicasthma. Inhibiting the influx of inflammatory cells, throughdown-regulation of ICAM-1 and suppression of the innate immune responsethrough inhibition of TLR-9 is an effective therapeutic approachprovided by the present invention. This was a surprising discovery asseveral studies have identified TLR9 agonists as a potential therapy forasthma. Several clinical trials have explored the use of TLR9 agoniststo treat asthma, for example, Astra Zeneca have a TLR9 agonist AZD1419,in-licensed from Dynavax, in Phase 2a for patients with eosinophilic,moderate to severe asthma via inhaled route. The TLR9 agonist suppressesTh2 (late phase) responses and enhanced Th1 responses. CytosBiotechnology has run clinical trials using the TLR-9 agonistCYT003-QbG10 to treat allergic bronchial asthma. Other TLR9 agonistswhich have been evaluated in the clinic for treating asthma were AIC(Dynavax), AVE0675 and SAR-21609 (Sanofi-Aventis/Coley Pharmaceuticals);QAX-935 (Idera Pharmaceuticals/Novartis).

Eosinophilic sinusitis (ES) is an inflammation of the nasal mucosacharacterized by the chronic influx of eosinophils and other monocytesand lymphocytes from the blood into the nasal mucosa in response toenvironmental antigens. This reaction leads to a variety of symptomsincluding nasal discharge (rhinorrea), congestion, nasal polyps.Eosinophilic sinusitis includes sinusitis and rhinitis. A paper byLicari A et al., (International Journal of Immunopathology Pharmacology,2014 October-December; 27(4):499-508) explains how the upper and lowerairways may be considered as a unique entity, interconnected bycoexisting inflammatory processes that share common etiopathogenicmechanisms. The paper explains how previous studies have stronglydemonstrated a relationship between rhinosinusitis and asthma. This hasled to the introduction of the concept of ‘United Airways’, which hasalso been included in the WHO document Allergic Rhinitis and its Impacton Asthma (ARIA); this concept has important consequences also on thetreatment of these disorders. The present invention is used to treateosinophilic sinusitis.

Graft versus host disease occurs subsequent to bone marrowtransplantation performed for treatment of blood cell cancers such asleukemia. During transplantation, the host blood-cell forming organs areeliminated by radiation treatment and the donor (graft) marrowtransplanted to re-establish the organ's function. While this procedurecan effectively treat the cancer, the transplanted immune system canbegin to “reject” the host tissues. Sensitive tissues include the liver,skin, lungs and the GI tract. Systemic immunosuppression is used tocontrol the rejection in most of these tissues, but the GI mucosa isparticularly difficult to treat with systemic therapies. Inflammationoccurring in the small intestine and colon is better treated withtargeted therapy delivered directly to the gut lumen. The presentinvention in either an oral formulation or rectal enema formulation isused to suppress graft versus host disease.

In a paper by Calcaterra et al, in the Journal of Immunology, (2008,181, 6132-6139) the authors demonstrate that inhibition of TLR9 may leadto the treatment of GVHD. The authors used C57BL/6 knockout mice todemonstrate that when TLR9 knockout mice were used as graft recipients,survival improved compared to wild type recipient mice. Mice weremyeloablative-irradiated and injected with 10⁷ bone marrow cells and4×10⁷ splenocytes obtained from full MHC major and minor Ag-disparateBALB/c donors. Recipient mice were monitored for clinical signs of GVHD,weight and survival. Interestingly those mice with a TLR4 knockout didnot show an improved survival versus wild type recipient mice. Allwild-type and TLR4−/− mice succumbed to severe acute GVHD within 60days, while TLR9−/− mice showed a significantly higher survival rate,with four of eight mice still alive at the end of the experiment. TheGVHD clinical score in TLR9−/− mice was also significantly lower thanthat in TLR 4−/− and C57B/6 mice and this correlated with reducedintestinal damage in the small intestine and to a lesser effect in thelarge bowel in TLR9−/− mice. Finally, at the end of the experiment, allTLR9−/− surviving mice achieved complete immune reconstitution, showed100% donor peripheral blood lymphocyte cells. The results in this paperdemonstrate the important role that TLR9 plays in the pathogenesis ofGVHD.

Inflammation of the eye can also be treated with the present invention.Such conditions are dry eye or Sjogren's disease where reduction in theproduction of tear fluid results in local inflammation of the ocularmucosa. Dry eye disease is a common complaint of ophthalmic patients.Unaddressed conditions of dry eye can lead to erosion and abrasion ofthe epithelial cell surface of the cornea, raising susceptibility toinfection. Progression of the disease can lead to ulceration of thecornea, even loss of sight. Disease and some physical conditions canpredispose individuals to dry eye disorder, including; allergies,diabetes, lacrimal gland deficiency, lupus, Parkinson's disease,Sjogren's syndrome, rheumatoid arthritis, rosacea, and others.Medications for other diseases may cause or exacerbate dry eyedisorders, including diuretics, antidepressants, allergy medications,birth control pills, decongestants and others. Age related changes mayinduce or exacerbate dry eye as well. Post-menopausal women experiencechanges in hormonal levels that can instigate or worsen dry eye andthyroid imbalances may cause similar changes. Finally aging itself cancause reduction in lipid production with resultant dry eye.

The present invention can be used for treating dry eyes, chronic dry eye(CDE) disease, or dry eye syndrome in a subject in need thereof.Subjects suffering from dry eyes, chronic dry eye (CDE) disease, or dryeye syndrome can be identified by any or a combination of diagnostic orprognostic assays known in the art. For example, typical symptoms of dryeyes, chronic dry eye (CDE) disease, or dry eye syndrome include, butare not limited to, symptoms such as, e.g., stinging, burning, orscratchy sensation in the eyes, stringy mucus in or around the eyes,increased eye irritation from smoke or wind, eye fatigue, sensitivity tolight, eye redness, sensation of foreign object in the eyes, difficultywearing contact lenses, periods of excessive tearing, swollen eyes, eyediscomfort, eye pain, and blurred vision (which worsens at the end ofthe day or after focusing for a prolonged period).

In some embodiments, dry eyes is diagnosed by the tear osmolarity test.The tear osmolarity test measures the number of solid particles in atear. The higher the tear osmolarity typically indicates that the tearhas less water and more particles, e.g., salts, proteins, lipids, andmucin. A tear osmolarity score of below 308 mOsms/L is normal, 308-320mOsms/L is mild dry eyes, 320-340 mOsms/L is moderate dry eyes, andabove 340 mOsms/L is severe dry eye. The present invention can be usedto treat mild dry eyes, moderate dry eyes, moderate to severe dry eyesand severe dry eyes.

Symptoms of severe dry eye may include, amongst others, conjunctivalinjection (hyperemia); such as bulbar conjunctival hyperemia, inferiortarsal conjunctival hyperemia, nasal bulbar conjunctival hyperemia; lidmargin hyperemia, central corneal staining and redness of the eye.

More specifically, treatment includes “therapeutic” and “prophylactic”and these types of treatment are to be considered in their broadestcontext. The term “therapeutic” does not necessarily imply that asubject is treated until total recovery. Similarly, “prophylactic” doesnot necessarily mean that the subject will not eventually contract adisease condition.

Accordingly, therapeutic and prophylactic treatment includesamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylactic” may be considered as reducing the severity or theonset of a particular condition. “Prophylactic” also includes preventingreoccurrence of a particular condition in a patient previously diagnosedwith the condition. “Therapeutic” may also reduce the severity of anexisting condition.

In summary, the present invention provides details describing that:

-   -   Mucosal inflammation can be more effectively treated by agents        that can target multiple pathways in the innate immune system.    -   Blocking the influx of inflammatory/immune cells (ICAM-1) and        blocking the activation of the innate immune response (TLR-9)        together allows both an acute and durable response.    -   The 20-base oligonucleotide SEQ ID NO:1 known to block ICAM-1,        exerts both an acute and durable response because it also        inhibits the activation of TLR-9.    -   The primary sequence of SEQ ID NO:1 does not predict its action        as a TLR-9 antagonist.    -   The specific primary sequence of SEQ ID NO:1 suggests possible        secondary structures that could influence TLR-9 activity, but        does not predict the most stable or optimal structures.    -   Conditions that influence the secondary structure of SEQ ID NO:1        also influence its activity as a TLR-9 antagonist.    -   It has been discovered that an oligonucleotide, with the primary        sequence of SEQ ID NO:1, when submitted to conditions that        optimize its secondary structure, is a more potent treatment        agent for mucosal inflammation than predicted by the primary        sequence alone.

TLRs are a key means by which the host recognizes and mounts an immuneresponse to foreign molecules. They also provide a mechanism by whichthe innate and adaptive immune responses are linked. Specifically, TLR-9recognizes bacterial and viral DNA through certain unmethylated CpGmotifs not present in mammalian DNA. Cells contained within the mucosacan therefore “sense” and respond to the presence of “foreign” DNA.Binding of such ligands activates the immune system to further respondto and remove the pathogen.

It is also known that synthetic oligonucleotides (ODN) containing CpGdinucleotide sequences can stimulate immune responses through the TLR-9pathway. In addition, the use of synthetic oligonucleotides has shownutility as inhibitors of inflammatory cytokines and these actions areknown to be mediated though inhibitory actions on TLR-9.

The published literature documenting the properties of DNAoligonucleotides required for binding to TLR-9 is extensive and hasfocused on both agonist and antagonist sequences. While the CpG motif isknown to be required for stimulatory activity, there are no canonicalsequences known to absolutely predict antagonist sequences. Certaininhibitors have been described previously in the art. In addition tothese triplet-containing inhibitory ODNs, several groups have reportedother specific DNA sequences that could inhibit TLR-9 mediatedactivation by CpG containing ODNs. These “inhibitory” or “suppressive”motifs are rich in poly “G” (e.g., “GGG”) or “GC” sequences, tend to bemethylated and are present in the DNA of mammals and certain viruses.Other inhibitory sequences have been identified as containing a “GGGG”motif within the sequences. Certain repetitive TTAGGG elements, presentat high frequency in mammalian telomeres, have been observed todown-regulate CpG-induced immune activation demonstrate that syntheticoligonucleotides containing the TTAGGG element mimic this activity andcould be effective in the prevention/treatment of certain Th1-dependentautoimmune diseases.

The secondary structure of ODNs has also been studied as a basis fordefining the DNA structure necessary for binding to TLR-9. However,there does not appear to be a secondary structure that absolutelyspecifies binding affinity, although sequence specific effects onstructure are observed to alter the ODN agonist or antagonist activity.

The surprising finding is that SEQ ID NO:1 is an antagonist of TLR-9 andis devoid of agonist activity. Further, the invention defines specificconditions that contribute to the predicted secondary structure of SEQID NO:1 and relate the secondary structure to TLR-9 antagonism.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of the application, where;

FIG. 1 is a dose response graph for SEQ ID NO:1 stimulation of TLR-9.

FIG. 2 is a dose response of TLR-9 inhibition by SEQ ID NO:1.

FIG. 3 is a graph showing SEQ ID NO:1 inhibition curves for ICAM-1 andTLR-9.

FIG. 4 is a thermodynamic predication of duplex stability for SEQ IDNO:1 and ODN2006 hetero duplex vs SEQ ID NO:1 homo duplex.

FIG. 5 is a graph showing the inhibition of TLR-9 activated by E. coligenomic DNA.

FIG. 6 shows: Upper—CD spectra of SEQ ID NO:1 in ddH20 at 4° C.Lower—spectra 20-95° C.

FIG. 7a shows a 190 nm peak intensity as a function of temperature.

FIG. 7b shows a 220 nm peak intensity as a function of temperature.

FIG. 8 shows the spectra of SEQ ID NO:1 in 5 mM spermine.

FIG. 9 shows a schematic of the experimental set up.

FIG. 10 shows IC50's for SEQ ID NO:1 inhibition of ODN2006 activation ofTLR-9.

FIG. 11 shows the Corneal Fluorescein Staining (CFS) evaluation ofalicaforsen (1 mM and 10 mM dose) in scopolamine dry eye mouse model atday 6 and day 10. Each circle represents one eye and the line is themean of the group. Two-way and one-way ANOVA Analyses were performedfollowed by Dunnett's Tests for multiple comparisons * p<0.05; **p<0.001.

FIG. 12 shows the evaluation of alicaforsen in Ovalbumin-Induced MurineModel of Allergic Asthma. One-way ANOVA followed by Dunnett's multiplecomparison test compared to vehicle control; *p<0.05; ** p<0.01.Student's unpaired, two-tailed T-test comparing 1 mM alicaforsen tovehicle control; #p<0.05.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides the following examples which are notlimiting.

EXAMPLES Example 1: TLR-9 Inhibitory Activity

SEQ ID NO:1 was screened for dose response activity to determinepossible EC50 for TLR9 activation at seven different doses (0.01, 0.05,0.1, 0.5, 1, 5 and 10 μM) in triplicate. Briefly, the TLR9/NF-kBluciferase reporter HEK 293 cell line (Abeomics, San Diego, Calif.) wasplated in 96-well white solid plates at 5×10⁴ cells per well for 16 h.Cells were treated with different doses of SEQ ID NO:1 as well as with20 ug/ml of CpG ODN-2006, a known agonist of TLR-9 in triplicate for 16h. Luciferase activity was then measured and analyzed.

As shown in FIG. 1, SEQ ID NO:1 was devoid of agonist activity up toconcentrations as high as 100 uM while a 1.0 uM dose of ODN2006 produceda 7.5-fold increase in TLR-9 induced gene activation.

SEQ ID NO:1 was next screened for dose responsive inhibition activity todetermine the IC50 against TLR9-mediated NF-kB induction at sevendifferent doses (0.1, 1, 5, 10, 25, 50 and 100 μM) in triplicate.Briefly, the TLR9/NF-kB luciferase reporter HEK 293 cell line was platedin 96-well white solid plates at 5×10⁴ cells per well for 16 h. Cellswere pretreated with different doses of SEQ ID NO:1 in triplicate for 1h. Cells were then treated with 20 ug/ml of CpG ODN2006 to activateTLR9. After 16 h, luciferase activity was measured and analyzed. Theseresults are shown in FIG. 2.

The activity of SEQ ID NO:1 as an antagonist of TLR-9 requires higherconcentrations than the inhibition of ICAM-1 expression. The comparativedose responses are shown in FIG. 3, in relation to the dose used in theenema formulation.

It is evident from the data of FIG. 3 that the therapeutic dose of SEQID NO:1 used for the treatment of IBD (659 uM) is sufficient to providetherapeutic levels of the drug for both mechanisms of action.

It was also necessary to rule out a direct inhibitory effect of SEQ IDNO:1 on the binding of the activator ODN2006. A screen of the twostructures using the Oligo Analyzer 3.1 program (Integrated DNATechnologies, Inc.) showed that alicaforsen was energetically morelikely to form a dimer with itself than with ODN2006. This comparison isshown in FIG. 4.

While the data of FIG. 4 indicate that SEQ ID NO:1 is more likely toform a homodimer than a heterodimer with ODN2006, additional experimentstested the antagonist activity of SEQ ID NO:1 in the presence of E. coligenomic DNA as the activator. In this assay, SEQ ID NO:1 remainedantagonistic to the activation of TLR-9 (FIG. 5).

Example 2: Prediction of Optimum Secondary Structure

The homodimer shown in FIG. 4 is the most energetically favorable duplexstructure for SEQ ID NO:1. Of interest was whether this secondarystructure may influence the TLR-9 inhibitory activity and under whichconditions the duplex was most stable

The predicted duplex structure is stabilized by 4 Watson-Crick typebonds flanked by G-A mismatches and another 2 G-C pairs. The CpG motifsremain unconstrained on the non-overlapping 3′ ends for each duplexmember.

SEQ ID NO:1 was therefore subjected to Circular Dichroism (CD)spectroscopy to gather information about the possible secondarystructure. CD of DNA can be utilized to detect all 3 major forms ofduplex structure (B, A and Z) and is sensitive to conditions thatdisrupt the structure such a heating. Of interest was the stability ofany secondary structure detected at or above physiologic temperatures.Shown in FIG. 6 is the CD spectra of sample SEQ ID NO:1 in ddH20 at 4C.The profile shows features characteristic of duplexes in the B-form ofDNA, namely a characteristic low intensity positive band at ˜280 nm, twolow intensity negative bands at ˜210 nm and ˜255 nm and an positiveintense band at ˜190 nm

The positive band at 190 nm was found to be sensitive to temperaturebetween 4° C. and 20° C. Further heating to 80° C. showed a loss of >85%of the signal strength. This background signal was achieved at 40° C.making it unlikely that the structure is stable to physiologicaltemperature under these conditions.

Additional thermal denaturation of the putative duplex was also studiedin the presence of Na⁺ at concentrations of 50 mM and 150 mM. Sodiumfluoride was used as Cl⁻ ions interfere with the CD spectra in this UVregion.

As can be seen in FIG. 7(a), the intensity of the 190 nm peak is morestable to heating under both 50 and 150 mM sodium conditions, retainingat least 50% of its structure to 40° C. In addition, the intensity ofthe 190 nm peak returned to pre-heated levels once the samples werecooled back to 20° C. This implies that the observed secondary structureis reversible after melting.

No significant changes in the negative peak at 210 nm were detected uponheating. However, the positive peak at 220 nm was found to respondsimilarly to that of the 190 nm peak. These data are shown in FIG. 7(b).

Additional experiments were conducted to test the influence of Mg⁺⁺ andpolyamines on the stability of the structure. Surprisingly, thepolyamine, spermine at 5 mM in the presence of 50 mM Na⁺ completelyabolished the secondary structure (FIG. 8).

This could be a function of the altered phosphate backbone of SEQ IDNO:1 which contains 20 sulfur substitutions in place of O— on eachphosphate group (phosphorothioate).

Example 3: Influence of Structure Changes on TLR-9 Antagonism

To test the effect of treatments known to stabilize and/or destabilizethe secondary structure of SEQ ID NO:1 a series of experiments exploredthe effect of heat, Na+, Mg+ and spermine on the TLR-9 antagonistactivity of SEQ ID NO:1. The samples and conditions of these tests arediagramed in FIG. 9.

Briefly, samples of SEQ ID NO:1 were dissolved in Tris buffer at pH 7.2including 50 mM NaCl, in the presence of either 15 mM Mg⁺⁺ or 5 mMspermine or both Mg⁺⁺ and spermine. One sample remained at roomtemperature while the remaining samples were heated to 90° C. for 3 minand then allowed to cool to room temperature. The samples were thendiluted to the indicated concentrations and incubated with the targetcells for 1 hr, prior to the addition of 1.0 uM ODN2006 to stimulateTLR-9. The results of the experiment are shown in FIG. 10.

It can be seen in FIG. 10 that conditions which were observed to alterthe secondary structure of SEQ ID NO:1 also influence its activity as aninhibitor of TLR-9. It is also noted that these conditions weremaintained during the heating and cooling step where they had anopportunity to influence the secondary structure of SEQ ID NO:1.However, once diluted into the cell culture media, the conditions wouldbe altered to those of the media and the temperature would revert to 37°C. The differences seen are therefore stable under normal physiologicalconditions.

This alteration of conditions, when the samples are incubated withcells, may be the reason that some activity is still observed in thespermine-treated samples, even though the CD spectra would indicate thatthe pre-incubation conditions removed the secondary structure of theduplex. Alternatively, the results seen with spermine could representremaining TLR-9 inhibitory activity of non-duplexed molecules(monomers).

Example 4: Treatment of Inflammation of the Eye With alicaforsen

To test the effect of SEQ ID NO:1 on the condition inflammation of theeye, the effect of two different concentrations of alicaforsen in amurine model of dry eye by scopolamine administration was investigated.

Dry Eye Murine Mouse Model

In a model of mice, the application of transdermal scopolamine patchesto the mid-tail is used to reduce aqueous tear production and thus mimiclacrimal gland insufficiency. The function of scopolamine is to induce apharmacologic blockade of cholinergic receptors in the lacrimal glandand therefore to decrease aqueous production. The desiccation isamplified by adding environmental stress. Animals are exposed to alow-humidity environment and constant airflow.

Experimental Method Animals Used in the Study:

-   -   Species: Mouse.    -   Strain: C57BL/6N (pigmented).    -   Age: Approximately 6-7 weeks (at the first day of induction).    -   Number/sex: 55 females (study 40; reserve 15).

Throughout the study, animals had free access to food and water. Theywere fed with a standard dry pellet diet. Tap water was available adlibitum from plastic bottles.

40 animals were included in this study. Animals were selected based ongood health and homogeneous body weight. Only healthy animals withoutvisible ocular defect (corneal opacity) were involved in this study.Animals were randomized into the study groups using a macro function inExcel® software on the basis of the mean of the corneal fluoresceinstaining scores from both eyes on Day 3.

Dry eye symptoms were induced in pigmented C57BL/6N mice by exposingthem to a controlled environment room (approximate relative humidity<25%, temperature 22° C.±2° C.), in a cage with an air-flow around 15L/min and transdermal scopolamine administration (0.5 mg/72 h) foreleven days.

Scopolamine Administration

Mice were treated with transdermal scopolamine administration (0.5 mg/72h; Scopoderm TTS®). The transdermal scopolamine patch was wrapped nearthe tail base of the mouse, secured with cellophane tape. Patches werereapplied every 48 hours.

Route and Method of Administration and Justification

Mice were randomized into 4 groups of 10 animals. The study was dividedinto 2 experimental sets with 5 animals of each group represented. Allmice were treated on day one and then treated for 10 days in totalaccording to the following regime:

-   -   Optimmune® group: two doses per day on Days 3 and 10 and three        doses per day on Day 4 to Day 9.    -   alicaforsen (10 mM and 1 mM) group: one dose per day.    -   Vehicle Group: one dose per day.

All test items, control items and comparator item were instilled in botheyes (5 μL per administration), using a micropipette.

Tear production and corneal defects were assessed at baseline, on Days3, 6 and 10 using phenol red thread (PRT) and corneal fluorescencestaining (CFS), respectively, for each animal of the 4 groups.

General Clinical Signs

Body Weights

The body weight of all animals was recorded.

General Appearance

Each day, the general clinical signs and the appearance of all animalswas observed.

Ocular Examinations

Two types of ocular examinations were conducted:

A) Measurement of aqueous tear production PRT test.

-   -   Tear production was measured with the PRT test (Zone-Quick,        FCI-Ophthalmics) on both eyes, before administration on Day 3        and at least one hour after the second treatment for the other        days of the study. The thread was placed in the lateral cantus        of the lateral conjunctival fornix for 30 seconds. The thread        wet by tears would turn red, indicated aqueous tear production.        This data was expressed in millimetres.

B) Corneal fluorescein staining (CFS)

-   -   At the different time points the measurement was performed        before administration on Day 3 and at least one hour after the        second treatment for the other days of the study. The eyes of        the animals from all groups were examined by slit-lamp        observation using blue light after 0.5% fluorescein eye drop        instillation (0.5 μL). Punctuate staining was recorded with the        standardized National Eye Institute (NEI) grading system giving        a 0-3 score to each of the 5 areas in which the corneas were        divided.

Results: Animal Behaviour and Body Weights

A slight loss in body weight was observed for the majority of animals ofall groups between Day 0 and Day 10 due to dry eye conditions.

alicaforsen (10 mM and 1 mM), Vehicle, and Optimmune® did not affect thebehaviour of the animals.

Prt Test:

On Day 3, the tear production decreased for all groups. The values ofuntreated group were stable until Day 10. These data showed a goodinduction of dry eye in this murine model.

CFS Test:

Results from the CFS are summarized in Table 1 below and in FIG. 11:

TABLE 1 Dry eye symptoms evaluation Corneal fluorescein staining score(mean ± SD) Treatment Day 6 Day 10 ALICAFORSEN 10.8 ± 2.0  9.7 ± 2.2 (10mM) ALICAFORSEN 10.5 ± 2.6 9.3 ± 3.1 (p = 0.0429) (1 mM) Vehicle (PBS)10.5 ± 2.3 11.3 ± 2.4 Optimmune ® 8.0 ± 2.3 (p = 0.0021) 8.5 ± 2.3 (p =0.0020) Note: Statistical analysis relative to Vehicle group with Dunn'smultiple comparison tests: p < 0.05.

The Vehicle group had dry eye symptoms from Day 3 to Day 10, showingthat this study is validated.

CFS Scores:

The group treated with Optimmune® showed corneal fluorescein stainingscores lower than the Vehicle group on Day 6 (p=0.0021) and Day 10(p=0.0020).

The groups treated with alicaforsen (1 mM and 10 mM) had similar cornealfluorescein staining scores to Vehicle group on Day 6.

The groups treated with alicaforsen (1 mM and 10 mM) showed cornealfluorescein staining scores lower than the Vehicle group on Day 10 andthe group treated with alicaforsen (1 mM) showed a significantdifference (p=0.0429) compared to vehicle. Under these experimentalconditions, multiple topical administrations of alicaforsen (1 mM and 10mM) were clinically well tolerated.

The Vehicle group had dry eye symptoms from Day 3 to Day 10, showingthat this study is validated.

Optimmune® 0.2% and alicaforsen (1 mM) groups showed a statisticallysignificant reduction in the dry eye symptoms as measured by cornealfluorescein staining

Example 5: Treatment of Asthma with Alicaforsen

To test the effect of alicaforsen on conditions such as asthma, anovalbumin (OVA)-induced allergic asthma mouse model was treated withalicaforsen.

Method:

Allergic asthma was modeled in female BALB/c mice by initialsensitization to OVA followed by subsequent intranasal challenge ofpurified OVA.

Mice were monitored throughout the study for changes in body weight andgeneral signs of sickness. Allergic response to OVA was measured byexamining bronchoalveolar lavage fluid (BALF) for inflammatory cellinflux and the presence of the inflammatory cytokine IL-13.

Additionally, animals in experimental groups were treated via intranasal(IN) administration of either alicaforsen, at 1 mM dosing concentration,control vehicle (PBS), or intraperitoneal (IP) dexamethasone as apositive anti-inflammatory control.

Test Materials Test Item(s)

Table 2 and 3 below detail the test items and test materials used duringthe study.

TABLE 2 Cat. Lot Storage Exp. Material Name No. No. ManufacturerCondition Date Positive Dexamethasone NDC P355990 APP 18-22° C. AprilControl 63323-165-05 Pharmaceuticals 2018 Sensitization/ OVA, endotoxin-LS003059 55P16242 Worthington  2-8° C. December Challenge free 2017

TABLE 3 Cat. Lot. Storage Exp. Material Name No. No. ManufacturerCondition Date Positive Dexamethasone NDC 63323-165-05 P355990 APP18-22° C. April Control Pharmaceuticals 2018 Senaitization/ OVA,endotoxin- LS003059 55P16242 Worthington  2-8° C. December Challengefree 2017 Aluminum Vac-alu-250 5295 Invivogen 18-22° C. May Hydroxidegel 2018 PBS 10010031 RN13F2486 Life RT February Technologies 2018Anesthesia Isofluorane NDC 66794-013-25 B57D16A Piramal 18-22° C. MarchItem Healthcare 2021 Multiplex kit Mouse MCYTOMAG-70K 2990274 Millipore 2-8° C. July Cytokine/Chemokine 2018 magnetic Bead panel

Dosing of alicaforsen at a concentration of 1 mM was prepared based on a0.878 drug content factor and MW of 6795.9 g/mol. Prepared solutionswere stored at 2-8° C. for the duration of the study.

1 mL dexamethasone stock solution (4 mg/mL) was added to 3 mL saline fora solution concentration of 1 mg/mL.

OVA/Alum for Sensitisation:

Chicken egg OVAlbumin (OVA) was dissolved in PBS to a concentration of 1mg/mL. 1 mg/mL OVA solution was diluted 1:1 in Alum adjuvant and storedat 2-8° C. overnight until use. Final dosing concentration was 100 μgOVA per 200 μL OVA/alum mixture.

OVA for Challenge:

1 mL sterile PBS was added to 10 mg OVA for a solution concentration of10 mg/mL.

0.35 mL OVA stock solution was added to 1.75 ml PBS for a solutionconcentration of 1.67 mg/mL. Final dosing concentration=50 μg OVA in 30μL.

Animals Used in the Study:

Species/Strain: Balb/c Gender: Female Total # of 55 Animals: Age: 7-8weeks of age at study initiation. Body Weight: Weight variation of theanimals at study initiation did not exceed ±20% of the mean weight.Animals Health: The health status of the animals used in this study wasexamined on arrival. All animals were in good health, were acclimatizedto laboratory conditions, and were used in the study. Acclimation: Atleast 72 hours. Housing: During acclimation and study, animals werehoused within a limited access rodent facility and kept in groups ofmaximum 5 mice. Mice were housed in sterilized individually ventilatedpolysulfone cages with irradiated cob bedding material. Food and Water:Animals were provided ad libitum a commercial rodent diet and freeaccess to drinking water, supplied to each cage via sterilisedpolyethylene bottles. All food and water is sterilised. Environment:Automatically controlled environmental conditions were set to maintaintemperature at 20-26° C. with relative humidity (RH) of 30-70%, a 12:12hour light:dark cycle. Randomisation: Animals were randomly assigned tocages on arrival. Animals were assigned to treatment groups on day −1.Termination: Euthanasia anesthesia overdose.

Test Groups

The table 4 below lists the experimental group(s) used in the study.

TABLE 4 Group Group Group Disease TI/vehicle TI/vehicle TI/vehicleTI/vehicle Number Size Description Induction Route Dose Level VolumeDosage Dosing Regime 1 N = 5 Naive N/A N/A N/A N/A N/A 2 N = 10 DiseaseOnly Sensitization which NA NA NA NA 3 N = 10 Vehicle OVA/Alum via ip INNA 30 μl Once daily on 4 N = 10 Dexamethasone injection on day 0 IP 10mg/kg 10 ml/kg study days 14, 5 N = 10 Alicaforsca and 14, and IN  1 mM30 μl 25-27. challenged with OVAin PBS intranasal on days 14, 25-27

Disease Induction (Groups 2-6):

OVA Sensitisation:

On days 0 and 14, each animal was administered an IP injection of 200 μLOVA/Alum emulsion containing 100 μg OVA.

OVA Challenge:

On days 14 and 25-27, each animal was administered an intranasalchallenge of 30 μL PBS containing 50 μg OVA.

Treatment with Test Item:

alicaforsen was administered at 1 mM concentration intra-nasally (IN) ina volume of 30 μL. Treatments were performed on the same days as OVAchallenge on Study Days 14 and 25-27 (4 total treatments). Treatmentswere administered 1 hour following OVA challenge.

Positive Control Treatment:

10 mg/kg dexamethasone was administered in 200 μL volume/animal(intraperitoneal) on Study Days 14 and 25-27 (4 total treatments).

Observations and Examinations: Clinical Signs:

Careful examinations were carried out daily. Observations for changes inskin, fur, eyes, mucus membranes, occurrence of secretions andexcretions and autonomic activity were included. Significant changes ingait, posture, and response to handling, or the presence of bizarrebehavior, tremors, convulsions, sleep and coma were recorded.

Body Weight:

Body weight of animals was determined shortly before the studycommencement and twice weekly thereafter.

Termination, Tissue Sampling and Subsequent Analyses:

On Day 28, all mice were euthanised via detamine+xylazine overdose andexsanguination.

BALF Collection and Analysis:

Bonchoalveolar lavage was performed on euthanised animals. Briefly, anangiocatheter was placed into trachea. 1 mL of PBS was instilled intothe lungs and allowed to flow back out into the syringe; the PBS wasthen instilled and removed again. The resultant Bronchoalveolar lavagefluid (BALF) was centrifuged at 500×g for 5 mins.

The non-cellular portion of the BALF was stored at −80° C. The levels ofIL-13 were analyzed by Luminex technology.

The cellular portion of the BALF was used to analyze the cell influx.The total number of leukocytes within the BALF and different cell typespresent were examined via flow cytometry.

Granulocytes: CD45⁺; Non-autofluorescent; Gr-1⁺Eosinophils: CD45⁺; Non-autofluorescent; Gr-1⁺; Siglec F⁺

Results:

Animals sensitized and challenged with OVA protein showed signs ofdisease at experiment termination, including significantly increasedalveolar influx of total leukocytes, granulocytes and eosinophils whencompared to naïve mice. Furthermore, diseased animals showedsignificantly increased levels of IL-13 in BALF.

Treatment with the positive control dexamethasone significantly reducedgranulocyte and eosinophil populations and IL-13 levels in the BALF,indicating a reduction in the allergic response to OVA.

Intranasal administration of alicaforsen (1 mM) resulted insignificantly lower levels of total leukocytes (CD45+ cells) in the BALFand significantly reduced the proportion of eosinophils. There was alsoa significant reduction in total granulocyte frequency in 1 mMalicaforsen treated group compared to the vehicle group. Additionally,treatment with 1 mM alicaforsen led to significantly lower IL-13 levelsin the BALF. These data indicated a reduction in allergic response toOVA.

The results are shown in FIG. 12 and in Table 5 below, the resultsdemonstrate the anti-inflammatory effect of alicaforsen on a murinemodel of allergic asthma.

TABLE 5 Flow analysis of BALF mean, standard error of the mean (SEM) andIL-13 expression in BALF mean (SEM). Granulocyte Eosinophil (Frequencyof CD45+) (Frequency of CD45+) Mean SEM Mean SEM Group 1: Naive 37.14**8.17 5.16** 2.36 Group 2: Disease 84.23 0.92 76.23 1.63 only Group 3:Vehicle 86.69 1.22 81.97 1.69 Group 4: 51.88** 8.53 46.60** 8.70Dexamethasone Group 5: 80.16^(#) 2.56 56.64**^(##) 8.02 alicaforsen 1 mMTotal CD45 BALF cells in BALF IL-13 pg/ml Mean SEM Mean SEM Group 1:Naive   20186** 1095 8.0** 0.0 Group 2: Disease 413776 57945 53.8 11.8only Group 3: Vehicle 521708 51727 27.5 5.5 Group 4: 236574 63225 13.2**2.3 Dexamethasone Group 5:    217303**^(##) 26340 13.4*^(#) 2.6alicaforsen 1 mM

1. A pharmaceutical composition comprising SEQ ID NO:1 with a cation,optionally selected from the following list Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, Ba⁺⁺,Mn⁺⁺, Ni⁺⁺, Li⁺⁺, Zn⁺⁺, and Cr⁺⁺⁺, preferably Na⁺, more preferably40-200 mM Na⁺, optionally including 2-20 mM Mg²⁺.
 2. A composition asclaimed in claim 1, wherein the Na⁺ is 100-190 mM, more preferably140-160 mM.
 3. A composition as claimed in any one of claims 1 to 2,which also comprises one or more of hydroxymethyl cellulose, methylparaben sodium, propylparaben sodium, monobasic sodium phosphatemonohydrate, sodium hydroxide, hydrochloric acid and/or water.
 4. Acomposition as claimed in any one of claims 1 to 3 wherein thecomposition is in the form of a liquid syrup, gel, film, cream, powder,tablet and/or enema.
 5. A composition as claimed in any one of claims 1to 4, wherein the components are in the following ranges SEQ ID NO: 1 4mg hydroxymethyl cellulose 7-8 mg, optionally 7.5 mg methylparabensodium 16.6 mM 2.8-3.0 mg propylparaben sodium 1.4 mM 0.28-3 mgmonobasic sodium phosphate 4.4-4.6 mg monohydrate37.5 mM


6. A composition as claimed in any one of claims 1 to 5 for use inmedicine.
 7. A composition, as claimed in claim 6, wherein the use isfor the prevention or treatment of inflammatory bowel disease, rectalstump, radiation-induced proctitis, pouchitis, asthma, inflammation ofthe eye, dry eye, rhinitis, sinusitis or graft versus host disease. 8.Use of SEQ ID NO:1 in the manufacture of a medicament as claimed in anyone of claims 1 to 6 for the prevention or treatment of inflammatorybowel disease, rectal stump, radiation-induced proctitis, pouchitis,asthma, inflammation of the eye, dry eye, graft versus host disease,rhinitis or sinusitis.
 9. A method of treating inflammatory boweldisease, rectal stump, radiation-induced proctitis, pouchitis, asthma,inflammation of the eye, dry eye, rhinitis or sinusitis, or grant-versushost disease, the method comprising administration of a composition asclaimed in any one of claims 1 to 5 to a patient in need thereof.
 10. Amethod of making a composition as claimed in any one of claims 1 to 5,the method comprising combining SEQ ID NO:1 with a cation, optionallyselected from the following list Na⁺, K⁺, Mg⁺⁺, Ca⁺⁺, Ba⁺⁺, Mn⁺⁺, Ni⁺⁺,Li⁺⁺, Zn⁺⁺, and Cr⁺⁺, preferably Na⁺, more preferably 40-200 mM Na⁺,optionally including 2-20 mM Mg²⁺.
 11. A composition, as claimed in anyone of claims 1 to 6, wherein the composition is in the form of an enemaand wherein the composition is formulated in a dosage form to provide aconcentration of SEQ ID NO:1 at 2 mg/ml per day.
 12. A composition, asclaimed in any one of claims 1 to 6, wherein the composition is in theform of an enema and wherein the composition is formulated in a dosageform to provide a concentration of SEQ ID NO:1 at 1-4 mg/ml per day